Cloning of IgE-binding proteins from Simulium vittatum and their potential significance as allergens for equine insect bite hypersensitivity

Insect bite hypersensitivity (IBH) is an allergic dermatitis of horses caused by bites of Culicoides and sometimes Simulium spp. The aim of this investigation was to identify Simulium allergens associated with IBH. A phage surface display cDNA library expressing recombinant Simulium vittatum salivary gland proteins was screened using sera of IBH-affected horses sensitized to S. vittatum salivary gland proteins as shown in immunoblot, resulting in the identification of seven cDNAs encoding IgE-binding proteins. The deduced amino acid sequences of these proteins showed sequence similarities to antigen 5 like protein (Sim v 1), to a serine protease inhibitor (Sim v 2), to two alpha-amylases (Sim v 3 and Sim v 4), and to three S. vittatum erythema proteins (SVEPs). The cDNA inserts were subcloned and expressed as [His](6)-tagged protein in Escherichia coli and purified using Ni(2+)-chelate affinity chromatography. Mice were immunised with the seven recombinant proteins and the antibodies tested against the recombinant proteins and salivary gland extract (SGE) of S. vittatum and Culicoides nubeculosus in immunoblot analyses. r-Sim v 1 specific mouse Abs recognized a band of about 32 kDa in immunoblots of both S. vittatum and C. nubeculosus SGE, detectable also by serum IgE of IBH-affected horses. Preincubation of horse serum with r-Sim v 1 completely inhibited IgE binding to the 32 kDa band demonstrating the presence of cross-reactive antigen 5 like proteins in both SGE. Determination of IgE levels against the r-Sim v proteins and crude S. vittatum extract by ELISA in sera from 25 IBH-affected and 20 control horses showed that IBH-affected horses had significantly higher IgE levels than controls against r-Sim v 1, 2, 3, 4 and S. vittatum extract, whereas the r-SVEP showed only marginal IgE binding. Further analyses showed that 60% of IBH-affected horses reacted to r-Sim v 1, suggesting that this could be a major allergen for IBH. Forty to twenty percent of the IBH-affected horses reacted with r-Sim v 2, 3 or 4. Combination of the results obtained with the 4 r-Sim v proteins showed that 92% of the IBH-affected but only 15% of the healthy horses had IgE levels against one or more of the 4 r-Sim v proteins. Seventy percent of the healthy horses had detectable IgE against S. vittatum extract, indicating a low specificity of the detection system used. Optimization of the ELISA system will be required to determine reliable cut-off values for the IBH-related allergens. Their in vivo relevance needs to be carefully assessed.

The cDNA cloning of murine HIP /L29 and the functional analysis of HIP/L29 domains

Human heparin/heparan sulfate interacting protein/L29 (HIP/L29) is a heparin/heparan sulfate (Hp/HS) binding protein found in many adult human tissues. Potential functions of this protein are promotion of embryo adhesion, modulation of blood coagulation, and control of cell growth. While these activities are diverse, the ability of human HIP/L29 to interact with Hp/HS at the cell surface may be a unifying mechanism of action since Hp/HS influences all of these processes. A murine ortholog has been identified that has 78.8% homology over the entire sequence and identity over the N-terminal 64 amino acids when compared to human HIP/L29. Northern, Western, and immunohistochemical analysis shows that murine HIP/L29 mRNA and protein are expressed in a tissue specific manner. Murine HIP/L29 is enriched in the membrane fraction of NmuMG cells where it is eluted with high salt, suggesting that it is a peripheral membrane protein. The ability of murine HIP/L29 to bind Hp is verified by studies using native and recombinant forms of murine HIP/L29. A synthetic peptide (HIP peptide-2) derived from the identical N-terminal region of HIP/L29 proteins was tested for the ability to bind Hp and support cell adhesion. This peptide was chosen because it conforms to a proposed consensus sequence for Hp/HS binding peptides. HIP peptide-2 binds Hp in a dose-dependent, saturable, and selective manner and supports Hp-dependent cell adhesion. However, a scrambled form of this peptide displayed similar activities indicating a lack of peptide sequence specificity required for activity. Lastly, an unbiased approach was used to identify sequences within human and mouse HIP/L29 proteins necessary for Hp/HS binding. A panel of recombinant proteins was made that collectively are deficient in every human HIP/L29 domain. The activities of these deletion mutants and recombinant murine HIP/L29 were compared to the activity of recombinant human HIP/L29 in a number of assays designed to look at differences in the ability to bind Hp/HS. These studies suggest that each domain within human HIP/L29 is important for binding to Hp/HS and divergences in the C-terminus of human and mouse HIP/L29 account for a decrease in murine HIP/L29 affinity for Hp/HS. It is apparent that multiple domains within human and mouse HIP/L29 contribute to the function of Hp/HS binding. The interaction of multiple HIP/L29 domains with Hp/HS will influence the biological activity of HIP/L29 proteins. ^

Molecular cloning of a family of xenobiotic-inducible drosophilid cytochrome P450s: Evidence for involvement in host-plant allelochemical resistance

Cytochrome P450s constitute a superfamily of genes encoding mostly microsomal hemoproteins that play a dominant role in the metabolism of a wide variety of both endogenous and foreign compounds. In insects, xenobiotic metabolism (i.e., metabolism of insecticides and toxic natural plant compounds) is known to involve members of the CYP6 family of cytochrome P450s. Use of a 3′ RACE (rapid amplification of cDNA ends) strategy with a degenerate primer based on the conserved cytochrome P450 heme-binding decapeptide loop resulted in the amplification of four cDNA sequences representing another family of cytochrome P450 genes (CYP28) from two species of isoquinoline alkaloid-resistant Drosophila and the cosmopolitan species Drosophila hydei. The CYP28 family forms a monophyletic clade with strong regional homologies to the vertebrate CYP3 family and the insect CYP6 family (both of which are involved in xenobiotic metabolism) and to the insect CYP9 family (of unknown function). Induction of mRNA levels for three of the CYP28 cytochrome P450s by toxic host-plant allelochemicals (up to 11.5-fold) and phenobarbital (up to 49-fold) corroborates previous in vitro metabolism studies and suggests a potentially important role for the CYP28 family in determining patterns of insect–host-plant relationships through xenobiotic detoxification.

Cloning of the cDNA for the TATA-binding protein-associated factorII170 subunit of transcription factor B-TFIID reveals homology to global transcription regulators in yeast and Drosophila

The human transcription factor B-TFIID is comprised of TATA-binding protein (TBP) in complex with one TBP-associated factor (TAF) of 170 kDa. We report the isolation of the cDNA for TAFII170. By cofractionation and coprecipitation experiments, we show that the protein encoded by the cDNA encodes the TAF subunit of B-TFIID. Recombinant TAFII170 has (d)ATPase activity. Inspection of its primary structure reveals a striking homology with genes of other organisms, yeast MOT1, and Drosophila moira, which belongs to the Trithorax group. Both homologs were isolated in genetic screens as global regulators of pol II transcription. This supports our classification of B-TFIID as a pol II transcription factor and suggests that specific TBP–TAF complexes perform distinct functions during development.

An ethylene-induced cDNA encoding a lipase expressed at the onset of senescence

A cDNA clone encoding a lipase (lipolytic acyl hydrolase) expressed at the onset of petal senescence has been isolated by screening a cDNA expression library prepared from carnation flowers (Dianthus caryophyllus). The cDNA contains the lipase consensus sequence, ITFAGHSLGA, and encodes a 447-amino acid polypeptide with a calculated molecular mass of 50.2 kDa that appears to be a cytosolic protein. Over-expression of the clone in Escherichia coli yielded a protein of the expected molecular weight that proved capable of deesterifying fatty acids from p-nitrophenylpalmitate, tri-linolein, soybean phospholipid, and Tween in both in vitro and in situ assays of enzyme activity. The abundance of the lipase mRNA increases just as carnation flowers begin to senesce, and expression of the gene is also induced by treatment with ethylene. Southern blot analyses of carnation genomic DNA have indicated that the lipase is a single copy gene. The lipase gene is also expressed in carnation leaves and is up-regulated when the leaves are treated with ethylene. Deesterification of membrane lipids and ensuing loss of membrane structural integrity are well established early events of plant senescence, and the expression pattern of this lipase gene together with the lipolytic activity of its cognate protein indicate that it plays a fundamentally central role in mediating the onset of senescence.

Molecular cloning of a cytochrome P450 taxane 10β-hydroxylase cDNA from Taxus and functional expression in yeast

The early steps in the biosynthesis of Taxol involve the cyclization of geranylgeranyl diphosphate to taxa-4(5),11(12)-diene followed by cytochrome P450-mediated hydroxylation at C5, acetylation of this intermediate, and a second cytochrome P450-dependent hydroxylation at C10 to yield taxadien-5α-acetoxy-10β-ol. Subsequent steps of the pathway involve additional cytochrome P450 catalyzed oxygenations and CoA-dependent acylations. The limited feasibility of reverse genetic cloning of cytochrome P450 oxygenases led to the use of Taxus cell cultures induced for Taxol production and the development of an approach based on differential display of mRNA-reverse transcription-PCR, which ultimately provided full-length forms of 13 unique but closely related cytochrome P450 sequences. Functional expression of these enzymes in yeast was monitored by in situ spectrophotometry coupled to in vivo screening of oxygenase activity by feeding taxoid substrates. This strategy yielded a family of taxoid-metabolizing enzymes and revealed the taxane 10β-hydroxylase as a 1494-bp cDNA that encodes a 498-residue cytochrome P450 capable of transforming taxadienyl acetate to the 10β-hydroxy derivative; the identity of this latter pathway intermediate was confirmed by chromatographic and spectrometric means. The 10β-hydroxylase represents the initial cytochrome P450 gene of Taxol biosynthesis to be isolated by an approach that should provide access to the remaining oxygenases of the pathway.

Purification and cDNA Cloning of Maize Poly(ADP)-Ribose Polymerase

Poly(ADP)-ribose polymerase (PADPRP) has been purified to apparent homogeneity from suspension cultures of the maize (Zea mays) callus line. The purified enzyme is a single polypeptide of approximately 115 kD, which appears to dimerize through an S-S linkage. The catalytic properties of the maize enzyme are very similar to those of its animal counterpart. The amino acid sequences of three tryptic peptides were obtained by microsequencing. Antibodies raised against peptides from maize PADPRP cross-reacted specifically with the maize enzyme but not with the enzyme from human cells, and vice versa. We have also characterized a 3.45-kb expressed-sequence-tag clone that contains a full-length cDNA for maize PADPRP. An open reading frame of 2943 bp within this clone encodes a protein of 980 amino acids. The deduced amino acid sequence of the maize PADPRP shows 40% to 42% identity and about 50% similarity to the known vertebrate PADPRP sequences. All important features of the modular structure of the PADPRP molecule, such as two zinc fingers, a putative nuclear localization signal, the automodification domain, and the NAD+-binding domain, are conserved in the maize enzyme. Northern-blot analysis indicated that the cDNA probe hybridizes to a message of about 4 kb.

Overexpression of an Arabidopsis cDNA Encoding a Sterol-C241-Methyltransferase in Tobacco Modifies the Ratio of 24-Methyl Cholesterol to Sitosterol and Is Associated with Growth Reduction

Higher plants synthesize 24-methyl sterols and 24-ethyl sterols in defined proportions. As a first step in investigating the physiological function of this balance, an Arabidopsis cDNA encoding an S-adenosyl-l-methionine 24-methylene lophenol-C241-methyltransferase, the typical plant enzyme responsible for the production of 24-ethyl sterols, was expressed in tobacco (Nicotiana tabacum L.) under the control of a constitutive promoter. Transgenic plants displayed a novel 24-alkyl-Δ5-sterol profile: the ratio of 24-methyl cholesterol to sitosterol, which is close to 1 in the wild type, decreased dramatically to values ranging from 0.01 to 0.31. In succeeding generations of transgenic tobacco, a high S-adenosyl-l-methionine 24-methylene lophenol-C241-methyltransferase enzyme activity and, consequently, a low ratio of 24-methyl cholesterol to sitosterol, was associated with reduced growth compared with the wild type. However, this new morphological phenotype appeared only below the threshold ratio of 24-methyl cholesterol to sitosterol of approximately 0.1. Because the size of cells was unchanged in small, transgenic plants, we hypothesize that a radical decrease of 24-methyl cholesterol and/or a concomitant increase of sitosterol would be responsible for a change in cell division through as-yet unknown mechanisms.

Cloning of a Tobacco Apoplasmic Invertase Inhibitor1 : Proof of Function of the Recombinant Protein and Expression Analysis during Plant Development

Higher plants express several isoforms of vacuolar and cell wall invertases (CWI), some of which are inactivated by inhibitory proteins at certain stages of plant development. We have purified an apoplasmic inhibitor (INH) of tobacco (Nicotiana tabacum) CWI to homogeneity. Based on sequences from tryptic fragments, we have isolated a full-length INH-encoding cDNA clone (Nt-inh1) via a reverse transcriptase-polymerase chain reaction. Southern-blot analysis revealed that INH is encoded by a single- or low-copy gene. Comparison with expressed sequence tag clones from Arabidopsis thaliana and Citrus unshiu indicated the presence of Nt-inh1-related proteins in other plants. The recombinant Nt-inh1-encoded protein inhibits CWI from tobacco and Chenopodium rubrum suspension-cultured cells and vacuolar invertase from tomato (Lycopersicon esculentum) fruit, whereas yeast invertase is not affected. However, only in the homologous system is the inhibition modulated by the concentration of Suc as previously shown for INH isolated from tobacco cells. Highly specific binding of INH to CWI could be shown by affinity chromatography of a total cell wall protein fraction on immobilized recombinant Nt-inh1 protein. RNA-blot analysis of relative transcript ratios for Nt-inh1 and CWI in different parts of adult tobacco plants revealed that the expression of both proteins is not always coordinate.

Expression cloning of Forssman glycolipid synthetase: a novel member of the histo-blood group ABO gene family.

A phenotypic cloning approach was used to isolate a canine cDNA encoding Forssman glycolipid synthetase (FS; UDP-GalNAc:globoside alpha-1,3-N-acetylgalactosaminyltransferase; EC 2.4.1.88). The deduced amino acid sequence of FS demonstrates extensive identity to three previously cloned glycosyltransferases, including the enzymes responsible for synthesis of histo-blood group A and B antigens. These three enzymes, like FS, catalyze the addition of either N-acetylgalactosamine (GalNAc) or galactose (Gal) in alpha-1,3-linkage to their respective substrates. Despite the high degree of sequence similarity among the transferases, we demonstrate that the FS cDNA encodes an enzyme capable of synthesizing Forssman glycolipid, and demonstrates no GalNAc or Gal transferase activity when closely related substrates are examined. Thus, the FS cDNA is a novel member of the histo-blood group ABO gene family that encodes glycosyltransferases with related but distinct substrate specificity. Cloning of the FS cDNA will allow a detailed dissection of the roles Forssman glycolipid plays in cellular differentiation, development, and malignant transformation.

A maize cDNA encoding a member of the retinoblastoma protein family: involvement in endoreduplication.

Retinoblastoma (RB-1) is a tumor suppressor gene that encodes a 105-kDa nuclear phosphoprotein. To date, RB genes have been isolated only from metazoans. We have isolated a cDNA from maize endosperm whose predicted protein product (ZmRb) shows homology to the "pocket" A and B domains of the Rb protein family. We found ZmRb behaves as a pocket protein based on its ability to specifically interact with oncoproteins encoded by DNA tumor viruses (E7, T-Ag, E1A). ZmRb can interact in vitro and in vivo with the replication-associated protein, RepA, encoded by the wheat dwarf virus. The maize Rb-related protein undergoes changes in level and phosphorylation state concomitant with endoreduplication, and it is phosphorylated in vitro by an S-phase kinase from endoreduplicating endosperm cells. Together, our results suggest that ZmRb is a representative of the pocket protein family and may play a role in cell cycle progression. Moreover, certain plant monopartite geminiviruses may operate similarly to mammalian DNA viruses, by targeting and inactivating the retinoblastoma protein, which otherwise induces G1 arrest.

Expression cloning of the Golgi CMP-sialic acid transporter.

Translocation of nucleotide sugars across the membrane of the Golgi apparatus is a prerequisite for the synthesis of complex carbohydrate structures. While specific transport systems for different nucleotide sugars have been identified biochemically in isolated microsomes and Golgi vesicles, none of these transport proteins has been characterized at the molecular level. Chinese hamster ovary (CHO) mutants of the complementation group Lec2 exhibit a strong reduction in sialylation of glycoproteins and glycolipids due to a defect in the CMP-sialic acid transport system. By complementation cloning in the mutant 6B2, belonging to the Lec2 complementation group, we were able to isolate a cDNA encoding the putative murine Golgi CMP-sialic acid transporter. The cloned cDNA encodes a highly hydrophobic, multiple membrane spanning protein of 36.4 kDa, with structural similarity to the recently cloned ammonium transporters. Transfection of a hemagglutinin-tagged fusion protein into the mutant 6B2 led to Golgi localization of the hemagglutinin epitope. Our results, together with the observation that the cloned gene shares structural similarities to other recently cloned transporter proteins, strongly suggest that the isolated cDNA encodes the CMP-sialic acid transporter.

Purification and molecular cloning of an inducible gram-negative bacteria-binding protein from the silkworm, Bombyx mori.

A 50-kDa hemolymph protein, having strong affinity to the cell wall of Gram(-) bacteria, was purified from the hemolymph of the silkworm, Bombyx mori. The cDNA encoding this Gram(-) bacteria-binding protein (GNBP) was isolated from an immunized silkworm fat body cDNA library and sequenced. Comparison of the deduced amino acid sequence with known sequences revealed that GNBP contained a region displaying significant homology to the putative catalytic region of a group of bacterial beta-1,3 glucanases and beta-1,3-1,4 glucanases. Silkworm GNBP was also shown to have amino acid sequence similarity to the vertebrate lipopolysaccharide receptor CD14 and was recognized specifically by a polygonal anti-CD14 antibody. Northern blot analysis showed that GNBP was constitutively expressed in fat body, as well as in cuticular epithelial cells of naive silkworms. Intense transcription was, however, rapidly induced following a cuticular or hemoceolien bacterial challenge. An mRNA that hybridized with GNBP cDNA was also found in the l(2)mbn immunocompetent Drosophila cell line. These observations suggest that GNBP is an inducible acute phase protein implicated in the immune response of the silkworm and perhaps other insects.